Mast cells differ between species. For example, it is now well established that the types of mast cells which exist in rodents are different from those in humans. See, for example, THE LUNG: Scientific Foundations, Raven Press, Ltd., New York, Ch. 3.4.11 (1991). Moreover, mast cell populations exist within the same species that differ in phenotype, biochemical properties, functional and pharmacological responses and ontogeny. These recognized differences in mast cells both between and within species are referred to as mast cell heterogeneity. See, for example, Irani et al., "Mast Cell Heterogeneity," Clinical and Experimental Allergy, Vol. 19, pp. 143-155 (1989). Because different mast cells exhibit different responses to pharmacological agents, it is not predictable that compounds claimed to be anti-allergic ("mast cell stabilizers") will have clinical utility in specific mast cell populations. The assumption that mast cells are a homogeneous population and that therefore experiments in rat mast cells would be predictive of those in human cells is known to be incorrect. Church, "Is Inhibition of Mast Cell Mediator Release Relevant to the Clinical Activity of Anti-Allergic Drugs?," Agents and Actions, Vol. 18, 3/4, 288-293, at 291 (1986).
Examples exist in the art in which mast cell stabilizing drugs inhibit only select populations of mast cells. Disodium cromoglycate is an anti-allergic drug whose local effects are believed to be due to inhibition of mast cell degranulation (Church, Agents and Actions, at 288). This drug was shown to inhibit rodent mast cell degranulation. In human trials, 100 .mu.M of the drug inhibited mast cells obtained from bronchoalveolar lavage fluid. In dispersed human lung mast cell preparations, 1000 .mu.M of the drug was required to inhibit only 25% to 33% of histamine release. Finally, histamine release from human skin mast cells was not inhibited at all by disodium cromoglycate. Pearce et al., "Effect of Disodium Cromoglycate on Antigen Evoked Histamine Release in Human Skin," Clinical Exp. Immunol., Vol. 17, 437-440 (1974); and Clegg et al., "Histamine Secretion from Human Skin Slices Induced by Anti-IgE and Artificial Secretagogues and the Effects of Sodium Cromoglycate and Salbutanol," Clin. Allergy, Vol. 15, 321-328 (1985). These data clearly indicate that classification of a drug as an anti-allergic does not predict that the drug possess inhibitory effects on all mast cell populations.
Mast cell heterogeneity makes it desirable to test drug compounds for human conjunctival mast cell stabilizing activity using human conjunctival mast cells. Schwartz et al. have previously reported a method for obtaining monodispersed cell suspensions of human skin mast cells, J. Immunol., Vol 138, 2611 (1987). This method uses an enzyme mixture composed of collagenase (30 mg), hyaluronidase (10 mg) and deoxyribonuclease I (3 mg) per digestion for tissue quantities up to 4 grams. These quantities of enzymes calculate to approximately 13000 U collagenase, 3500 U hyaluronidase and 6000 U deoxyribonuclease I per digestion. Experiments conducted using this dispersion method yield cell suspensions of initially low viability (&lt;50%) when applied to human conjunctival tissue. Enrichment of the cell suspension for mast cells using a 30% Percoll.RTM. cushion yielded about a 4 to 5 fold increase in the number of mast cells. These cells were still functional as assessed by calcium ionophore A.sub.23187 stimulation (40-80% of total histamine release was elicited with 10 .mu.M A.sub.23187). However, the spontaneous release of histamine from these preparations was unacceptably high (&gt;10% of total release) while the stimulated release of histamine using anti-human IgE (10 .mu.g/ml) was low (10 to 18% of total histamine release). Consequently, the treatment window between spontaneous and stimulated release was not sufficiently large enough to assess the activity of test compounds on histamine release from the mast cells.
Undem et al. have previously reported a method for obtaining monodispersed cell suspensions containing guinea pig lung mast cells. Am. Rev. Respir. Dis., Vol 133, 763-768 (1986). This method uses enzymes selected for the lung tissue but much lower concentrations of collagenase (125 U) per digestion per gram of tissue were employed. When concentrations of collagenase and hyaluronidase are adjusted down to concentrations more consistent with these levels, 200 U each per enzyme per digestion per gram of tissue, the viability of the post digestion populations increased to 70-80%. After enrichment using a 30% Percoll.RTM. cushion, the viabilities improved to &gt;95% though the numbers of mast cells were small (approximately 3%; typically&lt;100,000 mast cells). Cells obtained through the low concentration enzyme treatment were functional as assessed by calcium ionophore A.sub.23187 stimulation (45% of total histamine release was elicited with 10 .mu.M A.sub.23187). Spontaneous release of histamine was approximately 10% of total release. Anti-human IgE stimulated histamine release was improved to approximately 20% of total release but still was not sufficient to provide a large treatment window.
Additional treatment of the intact tissues from these digestions with collagenase and hyaluronidase at the much higher "Schwartz" levels (above) yielded greater numbers of mast cells (3-16 times greater than that already obtained through 200 U treatments). Viability of these cells post Percoll.RTM. enrichment was greater than 95%. Cells obtained with these additional treatments were functional as assessed by calcium ionophore A.sub.23187 stimulation (40% of total release was elicited with 10 .mu.M A.sub.23187). Moreover, these cells tend to yield lower spontaneous release levels (6%) but also lower total histamine release levels. Anti-human IgE stimulated histamine release was 15% of total release and still not sufficient to provide a large treatment window.
Accordingly, a need exists for an improved method of preparing human conjunctival mast cells for mast cell stabilization assays.